The invention relates to mutant CPB enzymes for use with prodrugs in ADEPT systems.
Targeting of drugs selectively to kill cancer cells in a patient has long been a problem for medical research. ADEPT is one approach to overcome the problem. ADEPT uses a tumour selective agent such as an antibody conjugated to an enzyme. The conjugate is administered to the patient (usually intravenously), allowed to localise at the tumour site(s) and clear from the general circulation. Subsequently a prodrug is administered to the patient which is converted by the enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an amplification effect is produced. Furthermore tumour cells not displaying the antigen recognised by the antibody (tumours usually display microheterogeneity) are also killed by enzymically amplified generation of the cytotoxic drug. A known system uses the procaryotic enzyme carboxypeptidase G2 (CPG2) as the enzyme component (see WO 88/07378).
A further problem with known systems is that repeated administration of the conjugate results in a host immune response rendering the therapy less effective. The antibody component is generally a mouse monclonal antibody which can be humanised using known techniques to reduce immunogenicity. However reduction of the immunogenicity of the enzyme component has proved more problematic. This is because the enzyme component must not be present naturally in the human host circulation otherwise premature conversion of prodrug to cytotoxic drug will occur and little selective toxicity to tumours will be observed.
These problems have been addressed in part by International Patent application WO 95/13095 (Wellcome Foundation). This application proposed ADEPT using mutant mammalian enzymes to activate prodrugs which are not activated by the corresponding native enzyme. However only ADEPT systems using mutants of carboxypeptidase A were enabled in the disclosure.
According to one aspect of the present invention there is provided a conjugate which is substantially non-immunogenic in humans comprising a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of a carboxypeptidase B (CPB) enzyme capable of converting a prodrug into an antineoplastic drug wherein the prodrug is not significantly convertible into antineoplastic drug in humans by natural unmutated enzyme.
Preferably the targeting moiety is an antibody.
Preferably the antibody is a F(abxe2x80x2)2 antibody fragment.
Preferably the enzyme is mutated to comprise a polarity change in its active site such that it can turn over a prodrug with a complementary polarity.
Preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having amino acid Asp 253 substituted by any one of Arg, Asn, Gln or Lys optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by any one of Arg, Lys or Asn;
natural amino acid Asp 145 substituted by any one of Val, Leu, Ile or Ala;
natural amino acid Ile 201 substituted by any one of Ser or Thr;
natural amino acid Ser 205 substituted by any one of Asn, Gln, His, Lys or Arg;
natural amino acid Ile 245 substituted by any one of Ser, Thr, Ala, Val, Leu, Asn, Gln, Lys, Arg or His;
natural amino acid Ala 248 substituted by any one of Asn, Gln, Lys, Arg, His, Ser or Thr;
natural amino acid Gly 251 substituted by any one of Thr, Asn, Ser, Gln, His, Lys, Arg, Val, Ile, Leu, Met, Phe, Ala or Norleucine; and
natural amino acid Cys 288 substituted by any one of Ser, Thr, Ala, Val, Leu or Ile.
More preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having natural amino acid Asp 253 substituted by any one of Arg or Lys and natural amino acid Gly 251 substituted by any one of Thr, Asn, Ser, Gln, Lys or Val, optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by Arg;
natural amino acid Asp 145 substituted by Ala;
natural amino acid Ile 201 substituted by Ser;
natural amino acid Ser 205 substituted by Asn;
natural amino acid Ile 245 substituted by any one of Ser, Ala or His;
natural amino acid Ala 248 substituted by any one of His, Ser or Asn; and
natural amino acid Cys 288 substituted by any one of Ser or Ala.
More preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having natural amino acid Asp 253 substituted by any one of Arg or Lys and natural amino acid Gly 251 substituted by any one of Thr, Asn or Ser optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by Arg;
natural amino acid Asp 145 substituted by Ala;
natural amino acid Ile 201 substituted by Ser;
natural amino acid Ser 205 substituted by Asn;
natural amino acid lie 245 substituted by Ala;
natural amino acid Ala 248 substituted by any one of Ser or Asn; and
natural amino acid Cys 288 substituted by Ser.
Especially the enzyme is any one of the following pancreatic human CPB mutants:
D253K; D253R; [G251N, D253K]; [G251T, D253K]; [G251S, D253K]; [G251T, D253R]; [A248S,G251T,D253K]; [A248N,G251N,D253K]; [A248S,G251N,D253K]; or [S205N,G251N,D253K].
According to another aspect of the present invention there is provided a matched two component system designed for use in a host in which the components comprise:
(i) a first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a CPB enzyme capable of converting a prodrug into an antineoplastic drug and;
(ii) a second component that is a prodrug convertible under the influence of the enzyme to the antineoplastic drug;
wherein:
the enzyme is a mutated form of a CPB enzyme;
the first component is substantially non-immunogenic in the host and; the prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme.
The term xe2x80x9cthe prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzymexe2x80x9d means that the prodrug does not give undue untargeted toxicity problems on administration to the host.
The term xe2x80x9csubstantially non-immunogenicxe2x80x9d means that the first component (conjugate) can be administered to the host on more than one occasion without causing significant host immune response as would be seen with for example the use of a mouse antibody linked to a bacterial enzyme in a human host.
Preferably the mutated enzyme is based on an enzyme from the same species as the host for which the system is intended for use but the mutated enzyme may be based on a host enzyme from a different species as long as the structure of the enzyme is sufficiently conserved between species so as not to create undue immunogenicity problems.
Preferably the targeting moiety is an antibody, especially an antibody fragment such as for example F(abxe2x80x2)2. Linkage to enzyme for conjugate synthesis may be effected by known methods such as use of heterobifunctional reagents as cross-linkers or by gene fusion or any other suitable method. Antibody may be from the same host (eg use of mouse antibody in mice) or the antibody may be manipulated such that it is not significantly recognised as foreign in the chosen host (eg use of chimeric, CDR grafted or veneered mouse antibodies in humans). Preferably the first component is a conjugate as defined above.
Transplantation of the variable domains of rodent antibodies into the constant domains of human antibodies (Chimeric antibodies) or building the antigen binding loops (CDRS) of rodent antibodies into a human antibody (CDR grafting) have both been shown to greatly decrease the immunogenicity of the rodent antibody in preclinical studies in monkeys and in patients. Even CDR grafted antibodies incorporate a large number ( greater than 50) of amino acids from the rodent antibody sequence into the human framework. Despite this in monkeys and patients greatly reduced immunogenicity has been reported. This provides evidence that mutating a very limited number of amino acids in the catalytic site of a host enzyme is likely to result in an enzyme with minimal immunogenicity and certainly lower immunogenicity than a non-host enzyme. The reader is directed to the following references: A. Mountain and J. R. Adair, Biotechnology and Genetic Engineering Reviews 10, 1-142, 1992; G. Winter and V. J. Harris, Trends in Pharmacological Sciences, 14, 139-143, 1993; I. I. Singer et al, J. Immunol, 150, 2844-57, 1993; J. Hakimi et al, J. Immunol, 147, 11352-59, 1991 and; J. D. Isacs et al, The Lancet, 340, 748-752, 1992. The constant region domains may be for example human IgA, IgE, IgG or IgM domains. Human IgG2 and 3 (especially IgG2) are preferred but IgG 1 and 4 isotypes may also be used. Human antibodies per se may also be used such as those generated in mice engineered to produce human antibodies. (Fishwald et al. in Nature Biotechnology (1996), 14, 845-851).
The host enzyme is mutated to give a change in mode of interaction between enzyme and prodrug in terms of recognition of substrate compared with the native host enzyme.
Preferably the enzyme mutation is a polarity change in its active site such that it turns over a prodrug with a complementary polarity; the prodrug not being significantly turned over by the unmutated host enzyme. Preferably the natural host enzyme recognises its natural substrate by an ion pair interaction and this interaction is reversed in the design of mutated enzyme and complementary prodrug. In this specification the term xe2x80x9cactive sitexe2x80x9d includes amino acid residues involved in any aspect of substrate recognition and/or catalytic functionality.
Point mutations will be referred to as follows: natural amino acid (using the 1 letter nomenclature), position, new amino acid. For example xe2x80x9cD253Kxe2x80x9d means that at position 253 of mature active HCPB an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one enzyme will be shown between square brackets with individual mutations separated by commas.
In this specification the term CPB includes the following:
i) mature, pro and prepro forms of the enzyme with or without xe2x80x9ctagsxe2x80x9d (eg c-myc);
ii) any carboxypeptidase with specificity for peptidic substrates having Lys or Arg at the C terminus having substantial sequence identity (preferably at least 60% identity, more preferably at least 70% identity, more preferably at least 80% identity and especially at least 90% identity) with mature active pancreatic HCPB within each of the key substrate binding sites 187-206 and 238-268;
preferably human pancreatic and plasma CPB enzymes (the pancreatic enzyme disclosed herein is preferred);
unless indicated otherwise or self evident from the context.
Naturally occurring allelic variants of CPBs are also contemplated. An allelic variant is an alternate form of sequence which may have a substitution, deletion or addition at one or more positions, which does not substantially alter the function of the CPB.
To determine the degree of identity between a carboxypeptidase and mature active pancreatic HCPB at its key substrate binding sites the following procedure is followed to align the sequences. When amino acid residues 109 to 415 of SEQ ID NO: 39 are renumbered 1 to 307, and aligned with other carboxypeptidases using a Clustal method with PAM250 residue weighting as described in the LASERGENE biocomputing software for MACINTOSH User""s Guide, A Manual for the LASERGENE system (2nd Edition, 1994, published by DNASTAR Inc., 1228 South Park Street, Madison, Wis. 53715, USA) the key zinc binding residues (at H66, E69 and H194), the key terminal-carboxy substrate binding residues (at R124, N141, R142, and Y246) and the catalytic residues (at R124, Y246 and E268) are essentially aligned. The key substrate recognition residue is deemed to be D253, with the substrate recognition pocket lying between the core xcex2-sheet (including residues 187 to 206) and the active-site surface loop and helix (residues 238 to 268). Residues 263-268 (within sequence 238-268) are beta strand, although they are part of the core beta sheet.
Mutant CPBs of the invention are mutants of any of the above CPBs having the desired property required for the invention. The following mutants of pancreatic HCPB are preferred: D253K, D253R and; especially [G251N,D253R]; corresponding mutations in other CPBs are also contemplated. Key mutation positions are also set out in the following table.
A mutant CPB of the invention may also comprise other xe2x80x9cconservativexe2x80x9d mutations (insertions, substitutions and/or deletions) that do not significantly alter the properties of the key mutation. For the purposes of this document a conservative amino acid substitution is a substitution whose probability of occurring in nature is greater than ten times the probability of that substitution occurring by chance (as defined by the computational methods described by Dayhoff et al, Atlas of Protein Sequence and Structure, 1971, page 95-96 and FIGS. 9-10) and as set out in the following table.
References on CPBs include the following: Folk J E in The Enzymes Vol III, Academic Press (1971), pg 57; Coll M et al (1991) EMBO Journal 10, 1-9; Eaton D L et al (1991) J Biol Chem 266, 21833-21838; Yamamoto K et al (1992) J Biol Chem 267, 2575-2581; U.S. Pat. No. 5,364,934 (Genentech) and; International Patent Application WO 95/14096 (Eli Lilly).
According to another aspect of the present invention there is provided a system as hereinbefore defined for use in a method of controlling the growth of neoplastic cells in a host in which the method comprises administration to said host an effective amount of a first component, allowing the first component to clear substantially from the general circulation, and administering an effective amount of a second component. Preferably the components are administered intravenously.
According to another aspect of the invention there is provided a method of treating neoplastic cells in a host in which the method comprises administration to said host an effective amount of a first component, allowing the first component to clear substantially from the general circulation, and administering an effective amount of a second component wherein the components form a two component system as defined herein. Preferably the components are administered intravenously.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising an effective tumour localising amount of a first component as hereinbefore defined and a pharmaceutically acceptable carrier or diluent. Preferably the composition is suitable for intravenous administration. Preferably the first component is supplied as a dry solid which is reconstituted before use with a suitable diluent.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising an effective antitumour amount of a second component as hereinbefore defined and a pharmaceutically acceptable carrier or diluent. Preferably the composition is suitable for intravenous administration. Preferably the second component is supplied as a dry solid which is reconstituted before use with a suitable diluent.
E.coli MSD 1646 containing pCG330 (also known as pICI1698) was deposited under the Budapest Treaty on Nov. 23rd 1994 with the National Collection of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, Scotland, United Kingdom AB2 1RY; the accession number is NCIMB 40694. NCIMB 40694 is another aspect of the present invention.
Antibody A5B7 was deposited as hybridoma deposit reference 93071411 under the Budapest Treaty on Jul. 14th 1993 at ECACC, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK. A humanised antibody A5B7 in the form of a F(abxe2x80x2)2 is preferred.
Antibody 806.077 was deposited as hybridoma deposit reference 96022936 under the Budapest Treaty on Feb. 29th 1996 at ECACC, PHLS centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK. Antibody 806.077 is an alternative anti-CEA antibody to A5B7 which is suitable for use in the present invention.
According to another aspect of the present invention there is provided a method of making a first component (conjugate) as herein described by linking:
a targeting moiety capable of binding with a tumour associated antigen and;
an enzyme capable of converting a prodrug into an antineoplastic drug wherein the enzyme is a mutated form of a host CPB enzyme. Linking may be effected by chemical or molecular biological techniques.
According to another aspect of the present invention there is provided a first component of the present invention.
According to another aspect of the present invention there is provided a polynucleotide sequence capable of encoding a first component (conjugate) of the present invention.
According to another aspect of the present invention there is provided a vector comprising a polynucleotide sequence capable of encoding a first component of the present invention.
According to another aspect of the present invention there is provided a cell comprising a vector or a polynucleotide sequence capable of encoding a first component of the present invention.
According to another aspect of the present invention there is provided a mutant CPB enzyme having the desired properties of the invention.
According to another aspect of the present invention there is provided a polynucleotide sequence capable of encoding a mutant CPB enzyme of the present invention. The present invention further relates to polynucleotides which hybridize to the polynucleotides encoding mutant CPBs if there is at least 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term xe2x80x9cstringent conditionsxe2x80x9d means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
According to another aspect of the present invention there is provided a vector comprising a polynucleotide sequence capable of encoding a mutant CPB enzyme of the present invention. The polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other plasmid or vector may be used as long they are replicable and viable in the host. The appropriate DNA sequence may be inserted into the vector by a variety of procedures.
In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E.coli. lac or trp, the phage lambda P.sub.L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E.coli. The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E.coli, Streptomyces, Salmonella Typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as NSO, CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by for example calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection (cationic lipid-mediated delivery of polynucleotides [Felgner et al. in Methods: A Companion to Methods in Enzymology (1993) 5, 67-75] or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). The skilled reader will be able to select the most appropriate method for a given host. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. Coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pAT153 and pBluescript. Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 xe2x80x9cbackbonexe2x80x9d sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the NSO, C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5xe2x80x2 flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Expression products are recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem., 244:917 (1969). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be nonglycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
Other systems of expression are also contemplated such as for example transgenic non-human mammals in which the gene of interest, preferably cut out from a vector and preferably in association with a mammary promoter to direct expressed protein into the animal""s milk, is introduced into the pronucleus of a mammalian zygote (usually by microinjection into one of the two nuclei (usually the male nucleus) in the pronucleus) and thereafter implanted into a foster mother. A proportion of the animals produced by the foster mother will carry and express the introduced gene which has integrated into a chromosome. Usually the integrated gene is passed on to offspring by conventional breeding thus allowing ready expansion of stock. Preferably the protein of interest is simply harvested from the milk of female transgenic animals. The reader is directed to the following publications: Simons et al. (1988), Bio/Technology 6:179-183; Wright et al. (1991) Bio/Technology 9:830-834; U.S. Pat. Nos. 4,873,191 and 5,322,775. Manipulation of mouse embryos is described in Hogan et al, xe2x80x9cManipulating the Mouse Embryo; A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory 1986.
Transgenic plant technology is also contemplated such as for example described in the following publications: Swain W. F. (1991) TIBTECH 9: 107-109; Ma J. K. C. et al (1994) Eur. J. Immunology 24: 131-138; Hiatt A. et al (1992) FEBS Letters 307:71-75; Hein M. B. et al (1991) Biotechnology Progress 7: 455-461; Duering K. (1990) Plant Molecular Biology 15: 281-294.
If desired, host genes can be inactivated or modified using standard procedures as outlined briefly below and as described for example in xe2x80x9cGene Targeting; A Practical Approachxe2x80x9d, IRL Press 1993. The target gene or portion of it is preferably cloned into a vector with a selection marker (such as Neo) inserted into the gene to disrupt its function. The vector is linearised then transformed (usually by electroporation) into embryonic stem (ES) cells (eg derived from a 129/Ola strain of mouse) and thereafter homologous recombination events take place in a proportion of the stem cells. The stem cells containing the gene disruption are expanded and injected into a blastocyst (such as for example from a C57BL/6J mouse) and implanted into a foster mother for development. Chimeric offspring can be identified by coat colour markers. Chimeras are bred to ascertain the contribution of the ES cells to the germ line by mating to mice with genetic markers which allow a distinction to be made between ES derived and host blastocyst derived gametes. Half of the ES cell derived gametes will carry the gene modification. Offspring are screened (eg by Southern blotting) to identify those with a gene disruption (about 50% of progeny). These selected offspring will be heterozygous and therefore can be bred with another heterozygote and homozygous offspring selected thereafter (about 25% of progeny). Transgenic animals with a gene knockout can be crossed with transgenic animals produced by known techniques such as microinjection of DNA into pronuclei, sphaeroplast fusion (Jakobovits et al. (1993) Nature 362 255-258) or lipid mediated transfection (Lamb et al. (1993) Nature Genetics 5 22-29) of ES cells to yield transgenic animals with an endogenous gene knockout and foreign gene replacement.
ES cells containing a targeted gene disruption can be further modified by transforming with the target gene sequence containing a specific alteration, which is preferably cloned into a vector and linearised prior to transformation. Following homologous recombination the altered gene is introduced into the genome. These embryonic stem cells can subsequently be used to create transgenics as described above.
The term xe2x80x9chost cellxe2x80x9d includes any procaryotic or eucaryotic cell suitable for expression technology such as for example bacteria, yeasts, plant cells and non-human mammalian zygotes, oocytes, blastocysts, embryonic stem cells and any other suitable cells for transgenic technology. If the context so permits the term xe2x80x9chost cellxe2x80x9d also includes a transgenic plant or non-human mammal developed from transformed non-human mammalian zygotes, oocytes, blastocysts, embryonic stem cells, plant cells and any other suitable cells for transgenic technology.
According to another aspect of the present invention there is provided a cell comprising a vector or a polynucleotide sequence capable of encoding a mutant CPB enzyme of the present invention. According to another aspect of the present invention there is provided a nucleotide sequence encoding a mature human pancreatic carboxypeptidase B defined in SEQ ID NO: 39 from position 109 onwards or a mutant thereof in which there is a cysteine residue encoded at position 243. This cysteine residue at position 243 in the cloned sequence is not observed in other published human pancreatic carboxypeptidase B sequences, as highlighted by Yamamoto et al, in the Journal of Biological Chemistry, v267, 2575-2581, 1992, where she shows a gap in her sequence following the position numbered 244, when aligned with other mammalian pancreatic carboxypeptidase B amino acid sequences (see discussion in Reference Example 6). Preferably the nucleotide sequence is in isolated form, that is to say at least partially purified from any naturally occurring form. Preferably the mutants are mutant CPB enzymes suitable for the present invention.
According to another aspect of the present invention there is provided a method of making human pancreatic carboxypeptidase B or a mutant thereof in which there is a cysteine residue encoded at position 243 comprising expression in a host cell of a nucleotide sequence encoding a mature human pancreatic carboxypeptidase B defined in SEQ ID NO: 39 from position 109 onwards or a mutant thereof in which there is a cysteine residue encoded at position 243.
According to another aspect of the present invention there is provided prodrugs of Formula 1 wherein:
w represents a direct bond or CH2 
R1 and R2 independently represent Cl, Br, I or xe2x80x94OSO2Me
R3 and R4 independently represent H, C1-3alkyl, C1-3alkoxy, F or Cl C4alkyl or
R5 and R6 independently represent H or C1-4alkyl or
R3 and R6 together can represent xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94 to form a bicyclic ring system optionally containing 1-3 heteroatoms selected from O, N and S
X is selected from
xe2x80x94CHR7CHR8xe2x80x94 where R7 and R8 are selected from H and C1-4alkyl optionally substituted with phenyl provided at least R7 or R8 is H;
xe2x80x94NHCHR9xe2x80x94
where R9 is selected from H;
the side chain of common amino acids including for example the side chain of Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val;
(CH2)nCONHR10 where n=1-3 and R10 is selected from C1-6alkyl, cyclopentyl, cyclohexyl and phenyl and each R10 listed hereinbefore is optionally substituted with halogen, C1-4alkyl or C1-4alkoxy;
xe2x80x94NHxe2x80x94N(R12)xe2x80x94 where R12 is selected from H and C1-4alkyl;
Y represents NH or O
Z is selected from
xe2x80x94(CH2)nxe2x80x94CO2H (n=1-4)
xe2x80x94CH2OCH2CO2H
xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94CO2H
xe2x80x94(CH2)ntetrazol-5yl (n=1-4)
xe2x80x94(CH2)nCONHSO2R11 (n=1-4) in which R11 is selected from C1-4alkyl
xe2x80x94(CH2)nSO2NH2 (n=1-4) and salts thereof.
According to another aspect of the present invention there is provided any one of the following compounds or a pharmaceutically acceptable salt thereof:
a) N-(4-{4-[bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanine;
b) N-[N-(4-{4-[bis-(2-chloroethyl)-amino]-3-methyl-phenoxy}-benzoyl)-L-alanine]-L-glutamic acid;
c) N-(4-{4-[bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanine; or
d) N-[N-(4-{4-bis-(2-chloroethyl)-amino]-phenoxy}-benzoyl)-L-alanine]-L-glutamic acid.
Compounds b) and d) are preferred prodrug second components of the invention. Compounds a) and c) are the corresponding drugs.
According to another aspect of the present invention there is provided a compound of Formula 1 or prodrugs b) or d) described above or a pharmaceutically acceptable salt thereof for use as a medicament.
According to another aspect of the present invention there is provided the compound of Formula 1 or prodrugs b) or d) described above or a pharmaceutically acceptable salt thereof for preparation of a a medicament for treatment of cancer (in combination with a first component of the invention).
In this specification the generic term xe2x80x9calkylxe2x80x9d includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as xe2x80x9cisopropylxe2x80x9d are specific for the branched-chain version only. An analogous convention applies to other generic terms.
It is to be understood that, insofar as certain of the compounds of Formula 1 may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of being a substrate for mutant CPBs of the invention. However in compounds of Formula 1, at the carbon atom having groups Y, Z and COOH attached, if there is a corresponding free amino acid then the carbon atom preferably has an L configuration in the corresponding free amino acid.
The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, substrate properties against mutant CPBs may be evaluated using the standard laboratory techniques.
A suitable pharmaceutically-acceptable salt of a basic compound of Formula 1 is, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric or maleic acid. In addition a suitable pharmaceutically-acceptable salt of an acidic compound of Formula 1 is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
The compounds of this invention may be utilized in compositions such as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like. The compounds of this invention can be adminstered to patients (animals and human) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. Although the dose will vary from patient to patient depending upon the nature and severity of disease, the patient""s weight, special diets then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize, the dosage range will generally be about 1 to 1000 mg. per patient per day which can be administered in single or multiple doses. Preferably, the dosage range will be about 2.5 to 250 mg. per patient per day; more preferably about 2.5 to 75 mg. per patient per day.
Naturally, these dose ranges can be adjusted on a unit basis as necessary to permit divided daily dosage and, as noted above, the dose will vary depending on the nature and severity of the disease, weight of patient, special diets and other factors.
Typically, these combinations can be formulated into pharmaceutical compositions and discussed below.
About 1 to 100 mg. of compound or mixture of compounds of Formula 1 or a physiologically acceptable salt thereof is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutically practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.
Illustrative of the adjuvants which can be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent such as corn starch, pregelatinized starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. When the dosage unitform is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the active substance in a vehicle such as water for injection, a naturally occuring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate or the like. Buffers, preservatives, antioxidants and the like can be incorporated as required.
A compound of the invention of Formula I, or a pharmaceutically-acceptable salt thereof, may be prepared by any process known to be applicable to the preparation of structurally-related compounds. Such procedures are provided as a further feature of the invention and are illustrated by the following representative examples in which variable groups have any of the meanings defined hereinbefore unless otherwise indicated. Where a synthesis of a particular compound is expanded upon below it will be appreciated that the general methodology can be applied to cover all compounds of the particular structure under discussion.
1. Compounds with W=Direct Bond
Such compounds may be prepared as outlined in FIG. 9. These prodrugs are cleaved by mutant CPB to liberate an intermediate which further collapses to release the corresponding phenol mustard (typical IC50=1.5 xcexcM).
Suitable reagents for steps a-d include:
(a) DCCI, HOBT or water soluble carbodiimide (EDCI) or isobutyl chloroformate/triethylamine;
(b) TFA (if P1=t-butyl, P2 is benzyl) or H2,Pd/C (if P1 is benzyl and P2=t-butyl);
(c) EDCI,DHAP,CHCl3,
(d) H2/Pd/C if P2=benzyl or TFA if t-butyl is used in the protection.
Compound 2 in FIG. 9
i) When Y=NH2 and P2 is a protecting group such as benzyl, and when Z is for example xe2x80x94(CH2)nxe2x80x94CO2H (n=1-4) then when n=1, dibenzyl L-aspartic acid is used; when n=2, L-glutamic acid dibenzyl ester is used and; when n=3, L-2-amino adipic acid dibenzyl ester is used.
ii) When Z is xe2x80x94(CH2)n-tetrazole: in the case of for example n=2, the sequence of reactions illustrated in FIG. 10 is used to generate the required dibenzyl protected intermediate from the known methyl ester. Suitable reagents for steps a-e include:
(a) Cs2CO3, PhCH2Br, DMF;
(b) 10% Pd/C, H2,BOC-O-BOC;
(c) NaOH, MeOH, H2O;
(d) Cs2CO3, PhCH2Br, DMF; isomers separated;
(e) HCl, ether, CH2Cl2 
iii) When Z is xe2x80x94(CH2)nCONHSO2R11 in the case of for example n=2 and R11=Me, the protected intermediate is made from N-BOC-xcex1-benzyl glutamic acid as illustrated in FIG. 11. Suitable reagents for steps a-b include:
(a) MeSO2NH2, DCCI, DMAP;
(b) HCl, EtOAc.
When Z=xe2x80x94(CH2)nSO2NH2 in the case of for example n=2, then L-2-amino-4-sulfamoylbutyric acid-benzyl ester, produced from L-2-amino-4-sulfamoylbutyric acid (Aldrich Chemical Company), is used.
v) Compounds where Y is OH are generated by established routes or by for example using compounds such as L-malic acid instead of the corresponding L-glutamic acid.
Compound 3 in FIG. 9
i) When X=xe2x80x94CH2CH2xe2x80x94 the intermediate can be made by reacting a compound illustrated as compound 2 in FIG. 9 with succinic anhydride to generate the half succinate ester where P1=H. Alternatively half esters of succinic acid can be used to couple to the above intermediate instead of using succinic anhydride.
ii) When X=xe2x80x94NHCH(R9)xe2x80x94 the prodrug is cleaved by mutant CPB to generate a compound of Formula 5 which is directly cytotoxic. For example when R9 =(CH2)2CONHxe2x80x94nC4H and R1=R2=Cl, R3=R4=R5=R6=H the cytotoxicity versus LoVo cells is about IC50=20 xcexcM.
To make compounds where X=NHCH(R9) conventional peptide coupling methodology is used as illustrated in FIG. 12. The intermediate is then treated with acid (eg HCl/ether) to form the free amine. Coupling to the phenol mustard is carried out as illustrated in FIG. 13. Suitable reagents for step a include:
1. para-nitrophenylchloroformate, triethylamine, chloroform
2. triethylamine, CH2Cl2 or;
1. COCl2/Quinoline, CH2Cl2 
2. triethylamine, CH2Cl2 
Compounds where W=CH2 and X=xe2x80x94NHxe2x80x94NH(R12)xe2x80x94
Such compounds may be synthesised as illustrated in FIG. 14. Suitable reagents for steps a-b include:
(a) BOCxe2x80x94N(R12)xe2x80x94NH2, EDAC, CH2Cl2; TFA, HCl/ETOAc
(b)
1. pyridine, CH2Cl2 
2. triethylamine, CH2Cl2 
The resulting product is then deprotected by standard methods.
When a pharmaceutically-acceptable salt of a compound of the formula I is required, it may be obtained, for example, by reaction of said compound with a suitable acid or base using a conventional procedure. When an optically active form of a compound of the formula I is required, it may be obtained by carrying out one of the aforesaid procedures using an optically active starting material, or by resolution of a racemic form of said compound using a conventional procedure.
Further uses of mutant CPBs of the invention include the following.
i) Carboxypeptidase enzymes may be used for the sequential removal of C-terminal amino acids from proteins and, following amino acid analysis of the residues released, can be used for determining the C-terminal amino acid sequence of proteins (R. P. Ambler, in: Methods in Enzymology, 1967, vol. X1, 436-445, Academic Press). The use of a mutant CPB possessing specificity for C-terminal aspartate and glutamate residues allows the use of these enzymes in extending the scope and ease of C-terminal analysis by carboxypeptidase digestion.
ii) Mutant enzymes may be used as enzyme labels in immunoassays. Product from substrate (prodrug) turnover may be detected by any suitable technique eg HPLC. Immunoassay techniques using enzymes as labels are described in A Practical Guide to ELISA by D. M. Kemeny, Pergamon Press 1991.
The invention will now be described by the following non-limiting Examples (with reference to the Reference Examples) in which:
(i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration;
(ii) operations were carried out at room temperature, that is in the range 18-25xc2x0 C. and under an atmosphere of an inert gas such as argon;
(iii) column chromatography (by the flash procedure) and medium pressure liquid chromatography (MPLC) were performed on Merck Kieselgel silica (Art. 9385) or Merck Lichroprep RP-18 (Art. 9303) reversed-phase silica obtained from E. Merck, Darmstadt, Germany;
(iv) yields are given for illustration only and are not necessarily the maximum attainable;
(v) the end-products of the Formula I have satisfactory microanalyses and their structures were confirmed by nuclear magnetic resonance (NMR) and mass spectral techniques; unless otherwise stated, CDCl3 solutions of the end-products of the Formula I were used for the determination of NMR spectral data, chemical shift values were measured on the delta scale; the following abbreviations have been used: s, singlet; d, doublet; t, triplet; m, multiplet;
(vi) intermediates were not generally fully characterised and purity was assessed by thin layer chromatographic, infra-red (IR) or NMR analysis;
(vii) melting points are uncorrected and were determined using a Mettler SP62 automatic melting point apparatus or an oil-bath apparatus; melting points for the end-products of the formula I were determined after crystallisation from a conventional organic solvent such as ethanol, methanol, acetone, ether or hexane, alone or in admixture and;
(viii) all temperatures are in xc2x0C.